SUMMARY The overall goal of Project 1 is to induce tolerance of porcine kidneys in baboons by co-transplantation (Tx) of vascularized thymus. To date, we have induced donor-specific T cell unresponsiveness and prevented anti- donor elicited antibodies, but have not yet achieved long-term, immunosuppression-free survival of xenograft kidneys. During the previous project period, we identified two major obstacles to this goal and developed strategies to overcome them. These are: (i) early loss of xeno-thymokidneys (TKs) due to activation of latent porcine CMV (pCMV); long-term graft survival was restored by elimination of pCMV through cesarean section of donors; and (ii) development of severe proteinuria; this problem could be delayed (but not prevented) by preventing SMPDL- 3b-dependent disruption of pig podocytes through treatment with Rituximab in the peri-Tx period. We also demonstrated upregulation of porcine CD80 on podocytes to be associated with xenograft nephropathy. Our preliminary data demonstrated that a combination therapy of Rituximab, which binds to porcine podocyte SMPDL- 3b and Belatacept, which inhibits CD80 activation, greatly minimized development of proteinuria and markedly prolonged survival (90, 93 and >155 days), with in vitro donor-specific unresponsiveness. The latest animal (now >155 days) developed new emigrant T cells from the porcine thymic graft. Based upon the data developed by this team (Projects 2, 3, and 4), we have designed innovative strategies that aim to completely eliminate proteinuria and permit total discontinuation of immunosuppression. In Aim 1, we will first study the mechanism responsible for persistent proteinuria in order to develop an appropriate strategy to eliminate it. On the basis of our preliminary data, we hypothesize that in addition to a SMPDL-3b pathway, CD47 and SIRP-alpha incompatibility between species may promote innate immune activation that culminates in podocyte activation, which causes upregulation of CD80 on podocytes in TKs. We will study mechanisms of podocyte destruction in vitro and optimize the current therapy in vivo, using hCD47 transgenic GalT-KO pig TK donors. In Aim 2, we will continue our efforts to achieve xenograft tolerance, attempting to improve the immune status of recipients by reconstitution of host- and donor-restricted protective T cell immunity. Studies in Project 3 demonstrated limitations in human T cell function and homeostasis following development in a pig thymus. We will address the hypothesis that these abnormalities will be corrected by adding recipient thymic epithelial cells (TECs) to the porcine thymus graft. We have recently succeeded in preparing hybrid thymic porcine grafts in which NHP TECs engrafted. We will assess the impact of using these hybrid thymi on immune function in baboons. In Aim 3, we will combine this strategy with durable mixed chimerism developed in Project 2. The combination of hybrid thymic grafting and durable mixed chimerism should induce tolerance of both innate and adaptive immune responses, including NK and B cells. The combined approach will also optimize functions of T cells, providing protective immunity against infections of both the recipient and the donor graft and of Tregs that protect both against residual donor-reactive T cells and against autoimmunity.